Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products

Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products View ORCID Profile Daniel Bergen , View ORCID Profile Òscar Puiggené , View ORCID Profile Esteban Marcellin , View ORCID Profile Robert E. Speight , View ORCID Profile Pablo I. Nikel , View ORCID Profile Birgitta E. Ebert doi: https://doi.org/10.1101/2025.02.17.638771 Daniel Bergen 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St. Lucia, 4072, Brisbane, Australia 2 Advanced Engineering Biology Future Science Platform , CSIRO, Dutton Park, 4102, Brisbane, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel Bergen Òscar Puiggené 3 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Òscar Puiggené Esteban Marcellin 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St. Lucia, 4072, Brisbane, Australia 4 Australian Research Centre (ARC) Centre of Excellence in Synthetic Biology (CoESB) Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Esteban Marcellin Robert E. Speight 2 Advanced Engineering Biology Future Science Platform , CSIRO, Dutton Park, 4102, Brisbane, Australia 4 Australian Research Centre (ARC) Centre of Excellence in Synthetic Biology (CoESB) Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert E. Speight Pablo I. Nikel 3 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pablo I. Nikel Birgitta E. Ebert 1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St. Lucia, 4072, Brisbane, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Birgitta E. Ebert For correspondence: birgitta.ebert{at}uq.edu.au Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Lignin is an underutilised global resource with significant potential for the production of chemicals that are currently derived from fossil resources. However, biotechnological lignin valorisation faces various challenges including its recalcitrance and the toxicity of aromatic intermediates, products, and by-products like formaldehyde. While biochemical production from lignin-derived monomers has been demonstrated by disrupting native lignin degradation pathways, this approach required co-feeding additional carbon sources such as glucose for growth. This dependence on additional carbon sources can create competition with the food industry and undermine the economic sustainability of the bioprocess. Here, we report growth of a protocatechuate production strain of Pseudomonas putida EM42 on the by-products from p -coumarate and ferulate valorisation, achieving carbon efficiencies of up to 78 %. Additional flux balance analysis identified C1 assimilation pathways, including two novel pathways, beneficial for the growth on the formaldehyde by-product from ferulate degradation leading to improved carbon utilisation. This study demonstrates how by-product utilisation from lignin conversion can eliminate the need for co-feeding additional carbon sources, thereby potentially improving the efficiency of lignin valorisation. Introduction Comprising 15-40 % of dry weight in terrestrial plants, lignin is the second most abundant biopolymer worldwide ( Ragauskas et al ., 2014 ). Despite its abundance, the heterogenous nature of lignin limits its use as a resource for chemical production. Chemically depolymerised lignin is a highly complex mixture of various aromatic compounds ( Becker & Wittmann, 2019 ; Rinaldi et al ., 2016 ), from which individual compounds are difficult to fractionate. Biotechnological processes are better suited to convert such heterogenous mixtures ( Kamimura et al ., 2017 ; Linger et al ., 2014 ; Weiland et al ., 2022 ). This process captures a range of lignin monomers and converts them into a single compound. This concept, called metabolic funnelling, allows for the targeted production of value-added products from complex feedstocks. Despite progress, current biological lignin valorisation processes face challenges, in particular its reliance on co-feeding other carbon sources for biomass formation, e.g. glucose or acetate ( Becker et al ., 2018 ; Johnson et al ., 2017 ; Kohlstedt et al ., 2018 ; Rouches et al ., 2021 ; Werner et al ., 2023 ). This dependency is particularly pronounced in strains engineered to accumulate valuable intermediates from aromatic metabolism, as these strains are unable to fully utilise aromatic substrates as carbon and energy sources. Supplementing additional carbon sources is effective in maintaining growth of these engineered microbial strains. However, apart from increasing production costs, incorporating edible carbon sources undermines the advantage of using lignin, which is inedible and does not compete with food supply chains. Competition with the food industry is especially of growing concern given the increasing global population. Efforts to achieve “glucose-free” lignin valorisation, where lignin monomer fractions (H-, G-, S- lignin monomers) are bifurcated into substrates for growth and precursors for production ( Almqvist et al ., 2021 ; Shinoda et al ., 2019 ), avoid co-substrate supplementation but minimise the carbon conservation of lignin monomers in target products. Previous studies discuss growth on the C2 by-product fraction from p -coumarate ( Fenster et al., 2022 ; Salvachúa et al ., 2018 ). However, as far as we are aware, no study has investigated whether the acetyl-CoA (C2) by-product fraction from lignin monomers, such as p- coumarate and ferulate, is sufficient as sole carbon source to sustain growth of lignin valorising production strains. C1 molecules, such as formaldehyde, also occur as by-products during aromatic degradation, e.g. methoxylated G- and S-lignin monomers ( Klein et al ., 2022 ). Considering that the C2 by-product fraction might be insufficient as sole carbon source for proper growth of the microbial host, as suggested by Salvachúa et al ., (2018) , enabling C1 assimilation might be a promising addition to support growth on G- and S-monomers (e.g., ferulate, vanillin, vanillate, syringate). Such endeavours could harness synthetic pathways primarily developed for the utilisation of CO 2 -derived molecules as carbon feed for biotechnological processes ( Bruinsma et al ., 2023 ; He et al ., 2020 ; Turlin et al ., 2022 ; Wenk et al ., 2022 ). This study explores the potential of using the C1 and C2 by-product fractions from the degradation pathways of p -coumarate and ferulate for growth and cell maintenance while conserving the core aromatic structure in the target product. Pseudomonas putida was chosen as production host due to its native lignin degradation pathways ( Harwood & Parales, 1996 ; Jiménez et al ., 2002 ; Nogales et al ., 2017 ). Furthermore, Pseudomonas possess high tolerance towards oxidative stress and cytotoxic compounds as exhibited by aromatic compounds such as lignin monomers ( Blank et al ., 2008 ; Ebert et al ., 2011 ; Nikel et al ., 2016 , 2021 ), while being genetically accessible with a variety of genetic engineering tools ( Neves et al ., 2020 , 2024 ; Nikel et al ., 2014 ; Volke et al ., 2021 , 2022 ). These characteristics make P. putida a promising host for industrial bioconversion ( Nikel & de Lorenzo, 2018 ; Poblete-Castro et al ., 2012 ; Weimer et al ., 2020 ). As a proof-of-concept, we focussed on protocatechuate, the first common intermediate of p -coumarate, ferulate, vanillate, and vanillin degradation in P. putida . It is an important platform chemical for the production of a variety of aromatic compounds conventionally derived from petrochemical resources ( Johnson et al ., 2019 ; Kohlstedt et al ., 2018 ; Notonier et al ., 2021 ; Werner et al ., 2023 ). This study reports the co-substrate independent growth and protocatechuate production that solely rely on acetyl-CoA from the enoyl-CoA hydratase reaction in the p -coumarate and the ferulate branches of the lignin degradation pathway in P. putida. Furthermore, we investigate in silico the potential of heterologous C1 assimilation pathways for valorisation of the G lignin monomer, ferulate. Materials and Methods Bacterial Strains and Growth Media Experiments were performed with P. putida EM42, a genome-reduced derivative of P. putida KT2440 ( Martínez-García et al ., 2014 ). Deletion of pcaHG in the EM42 strain was performed according to Volke et al . (2021) via homologous recombination of the I-SceI recognition site-containing suicide plasmid pSNW2-Δ pcaHG and subsequent curing with the I-SceI endonuclease expressing plasmid pQURE·6. Successful deletion mutants were identified via colony PCR. The plasmid for deletion of pcaHG was cloned in Escherichia coli DH5α λpir using USER cloning ( Bitinaite et al ., 2007 ). Generated strains, plasmids, and primer sequences are listed in the supplemental material (Supplementary Table S1). For microtiter growth experiments and protocatechuate production experiments, two precultures were run prior to the main culture. The first preculture was grown overnight in LB medium, followed by a second overnight preculture in de Bont (dB) minimal medium ( Hartmans et al ., 1989 ) supplemented with 50 mM sodium acetate, 50 mM p -coumarate, or 50 mM ferulate, respectively. For main cultures, dB minimal medium supplemented with sodium acetate, p -coumarate, or ferulate was used at concentrations specified in the results section. The pH of p -coumarate and ferulate stocks was adjusted to 7.0 by addition of sodium hydroxide and the solutions were sterilised via filtration. Cultures with dB minimal medium were always supplemented with 10 g L -1 MOPS buffer (pH 7.0). All shake flask experiments were performed at 30 °C and 250 rpm in a rotary shaker. Microtiter Growth Experiments Initial growth characterisation experiments were conducted using the Growth Profiler GP960 (EnzyScreen, Heemstede, The Netherlands). Per condition, 1 mL dB minimal medium was inoculated with an overnight preculture to an initial optical density at 600 nm (OD 600 ) of 0.01. The inoculated culture was split into three single wells (300 μL each) in 96-well microtiter plates. The cultures were shaken at 250 rpm and at 30 °C. The cycle time was set to 600 s, and the shutter time was 4 ms. Green pixel values (G value ) were obtained from photographed wells and converted to OD 600 values via a polynomial regression ( equation 1 ). The equation was parameterised using P. putida EM42 cultures grown in LB medium with OD 600 values ranging from 0.01 to 6.0 as determined by photometric analyses. Microbial Protocatechuate Production from p -Coumarate or Ferulate Production of protocatechuate and other intermediate products downstream of the enoyl-CoA hydratase reaction was evaluated in shake flask experiments. For this, 25 mL of dB minimal medium with 75 mM p -coumarate / ferulate were inoculated to an initial OD 600 of 0.01. Cultures were incubated at 250 rpm and 30 °C. For HPLC analysis, 0.5 mL cell suspension was withdrawn from the cultures throughout cultivation (see Figure 2 ) and centrifuged at 13,300 rpm for 1 min. 400 μL of the supernatant was transferred in a new tube and stored at -80 °C until further HPLC analysis. Quantification of Extracellular Aromatic Compounds Supernatant samples were thawed and centrifuged at 16,000 g at 4 °C for 5 min to remove any remaining cells and cell debris. The supernatants were diluted appropriately prior to analysis. Samples were transferred to HPLC glass vials with conical glass inserts. The UHPLC analysis was performed on a ThermoFisher Scientific Vanquish DUO UHPLC with a DAD detector (ThermoFisher Scientific, Parkville, VIC, Australia). The compounds were separated on a Waters Acquity HSS T3 column (100×2.1 mm, 1.8 µm particles) (Waters, North Ryde, NSW, Australia). The column oven was set to 40 °C. A gradient was run for 11 min from 95 % 1:1000 formic acid in MilliQ water (mobile phase A) to 100 % 1:1000 formic acid in acetonitrile (mobile phase B) followed by 3 min flushing with 100 % mobile phase B. Between samples the column was equilibrated with 1:20 mobile phase B in mobile phase A for 6 min. The flow rate was set to 0.4 mL · min -1 . Aromatic compounds were detected at 280 nm, 320 nm, and 360 nm. Quantification was based on a standard calibration curve derived from serial dilution of chemical standards. The limits of quantification were 0.7 μM – 3 mM. Acquired data were analysed with the Chromeleon software 7.4 (ThermoFisher Scientific). Model Curation and Metabolic Modelling A curated version of the most recent metabolic model of P. putida KT2440, i JN1463, ( Nogales et al ., 2020 ) was used. Reactions identified to enable growth on C1 compounds (CO 2 , formate) were set irreversible to prevent this growth phenotype nonnative to P. putida KT2440; these included reactions mediated by α-ketoglutarate dehydrogenase and phosphoribosylglycinamide formyltransferase (model reaction identifiers AKGDa and GARFT, respectively). Genes and corresponding reactions with pWW0 gene identifiers, originating from the TOL plasmid of P. putida mt-2, the progenitor strain of P. putida KT2440 ( Bagdasarian et al ., 1981 ) were removed, as well as Ni 2+ requirement for biomass synthesis. A list of all curation steps can be found in Table 1 . View this table: View inline View popup Download powerpoint Table 1: List of modifications for the curation of the genome-scale model i JN1463. Flux balance analysis (FBA) was performed with COBRApy ( Ebrahim et al ., 2013 ). Uptake rates for simulations of growth on p -coumarate or ferulate were set as indicated in the respective section. Gene deletions were simulated using the COBRApy function ‘model.genes.id.knock_out()’, which sets the flux boundaries of all reactions associated with the respective gene to 0 mmol · gCDW -1 · h -1 . The objective function for all simulations was set to maximise biomass formation. Results and Discussion In silico Evaluation of Growth on C2 By-Product Fraction Prior to in vivo experiments, growth on p -coumarate and ferulate was tested in silico using flux balance analysis (FBA). Simulations were run in COBRApy using a curated version of the published genome-scale model i JN1463 ( Nogales et al ., 2020 ). p- Coumarate and ferulate uptake rates were set to 4.5 mmol · g -1 · h -1 for FBA, based on reported uptake rates of a range of aromatic compounds ( Ravi et al ., 2017 ). Protocatechuate production was simulated by deactivating the protocatechuate dioxygenase reaction PCADYOX, which oxidises protocatechuate to 3-carboxy- cis -muconate ( Figure 1A ). This reaction is associated with the genes PP_4655 ( pcaH ) and PP_4656 ( pcaG ), hence its inactivation resembles a Δ pcaHG deletion in P. putida . The uptake rates for all other carbon sources were set to 0 mmol g CDW -1 · h -1 forcing the model to rely solely on the available acetyl-CoA resulting from the enoyl-CoA hydratase reactions (COCOAHA and FCOAHA). Download figure Open in new tab Figure 1: A Pathway for microbial conversion of p -coumarate/ferulate to protocatechuic acid in the Δ pcaHG mutant. The release of acetyl-CoA (from p -coumarate and ferulate) and formaldehyde (only from ferulate) is highlighted in orange and in blue, respectively. Subsequently, cleaved acetyl-CoA is the sole carbon source for biomass formation. Acetate conversion to acetyl-CoA is included for comparison. B Growth of EM42 (dashed line) and the Δ pcaHG deletion mutant (solid line) grown on different acetate concentrations. C Comparison of EM42 and the Δ pcaHG deletion mutant grown on different p- coumarate concentrations. D Comparison of the growth of the Δ pcaHG mutant on the by-product fractions from the degradation of p -coumarate (dashed lines) and ferulate (solid lines). The experimental error is included as the standard deviation of triplicate growth experiments and indicated by the shaded area. The predicted biomass formation rates ( Table 2 ) indicated that the amount of acetyl-CoA generated during conversion of p -coumarate or ferulate to protocatechuate is indeed sufficient for maintaining growth of a pcaHG deletion mutant incapable of further degrading protocatechuate as suggested by Salvachúa et al ., (2018) . As expected, the growth rates were highly decreased compared to the template model (-69 % for both substrates). Growth on ferulate led to a higher predicted growth rate than on p- coumarate (∼13 % for both cases, with and without deactivation of the protocatechuate dioxygenase). In contrast to p- coumarate, conversion of ferulate to protocatechuate produces formaldehyde in addition to acetyl-CoA ( Figure 1A ). While formaldehyde is not a carbon source of P. putida , full oxidation of this C1 compound via its formaldehyde and formate dehydrogenases provides additional NADH (2 molecules of NADH per molecule of formaldehyde; Roca et al ., 2009 ; Roca & Ramos, 2009 ; Turlin & Puiggené et al ., 2023; Zobel et al ., 2017 ). Though it has not been reported that additional NADH leads to improved growth rates in P. putida , the biomass yield on the main carbon source can be increased by providing auxiliary co-substrates like formate serving as an electron source ( Zobel et al ., 2017 ). This benefit is based on reduced rates of substrate oxidation in the central carbon metabolism due to metabolic redirection from catabolism towards anabolism, which preserves the carbon from the main carbon source in biomass ( Zobel et al ., 2017 ). In the simulations, NADH produced from formaldehyde oxidation reduced the oxidation of acetyl-CoA in catabolic reactions, thereby increasing the carbon available for anabolic processes and the biomass formation rate. View this table: View inline View popup Download powerpoint Table 2: Predicted maximal growth rates on ferulate and p -coumarate of the template model and the model simulating deletion of pcaHG with and without enforced protocatechuate (PCA) production. The simulation with p -coumarate as the substrate did not show protocatechuate production. Instead, conversion stopped at p -hydroxybenzoate, the immediate precursor of protocatechuate. This occurred due to the NADH demand for the conversion of p -hydroxybenzoate to protocatechuate. NADH formed in the previous step (the conversion of p -hydroxybenzaldehyde to p- hydroxybenzoate) could be reinvested here. However, since the simulations maximised growth, NADH is preferentially used for biomass formation, which is limiting in this scenario where only the acetyl-CoA by-product fraction of the p -coumarate branch is available. Enforcing protocatechuate production in the simulation with p -coumarate, lowered the biomass formation rate by 22 % ( Table 2 ). Similar to the formation of protocatechuate from p -hydroxybenzoate, the production of protocatechuate from vanillate in the ferulate branch requires one molecule of NADH per molecule of vanillate. However, the oxidation of the released formaldehyde provides additional reduced redox equivalents that outweigh the NADH demand required for vanillate conversion. Nevertheless, no complete conversion of vanillate to protocatechuate was observed in the simulations with protocatechuate formation rates reaching only 73 % of the ferulate uptake rate. However, this is a modelling artefact and not due to a lack of energy, as enforcement of protocatechuate production from ferulate did not lead to a reduction in biomass formation suggesting that conversion of vanillate to protocatechuate potentially leads to excess NADH. Indeed, the limitation by the last step in protocatechuate production from p -coumarate/ferulate as suggested by the model has previously been observed in vivo ( Kuatsjah et al ., 2022 ; Mohamed et al ., 2020 ; Werner et al ., 2023 ). In vivo Growth on C2 By-Product Fraction from p -Coumarate/Ferulate Degradation To confirm the in silico results, microtiter growth experiments with p -coumarate and ferulate were performed. Acetate was included as the control condition. The activation of acetate to acetyl-CoA requires the same co-factors as the activation of p -coumarate and ferulate ( Figure 1A ). The remaining two steps for protocatechuate production catalysed by vanillin dehydrogenase and p- hydroxybenzoate dehydrogenase, encoded by vnd and pob A respectively, are redox balanced. Therefore, they do not increase the net cofactor demand. Hence, growth on acetate should be similar to p -coumarate in a strain incapable of performing protocatechuate catabolism. Initial comparison of the growth behaviour of the parental strain P. putida EM42 (EM42) and the Δ pcaHG mutant on acetate revealed identical growth of both strains ( Figure 1B and Table 3 ) showing that the deletion of pcaHG does not affect acetate metabolism. On p -coumarate, EM42 Δ pcaHG reached a maximum optical density (OD max ) of only 9.6–12.4 % of the maximum OD 600 of the parental strain ( Figure 1C and Table 3 ). This was expected due to the smaller proportion of carbon available for growth. Unlike EM42, the pcaHG deletion mutant can only utilise the C2 by-product fraction (acetyl-CoA) lowering the available carbon pool by ∼80 %. View this table: View inline View popup Download powerpoint Table 3: Comparison of maximum growth rate (μ max ) and maximum optical density (OD max ) of EM42 and EM42 Δ pcaHG grown on different concentrations of acetate and p -coumarate. Errors are based on the standard deviation of biological triplicates. Despite the same cofactor demand, growth on acetyl-CoA derived from p- coumarate led to 60 % lower final optical densities compared to growth on acetate, and growth was also slowed by 40–60 %. These differences could be attributed to the toxicity of p -coumarate and its aromatic breakdown products ( Calero et al ., 2018 ; Mohamed et al ., 2020 ; Salvachúa et al ., 2018 ; Zhang et al ., 2021 ). Next, we compared growth of the pcaHG deletion mutant on p -coumarate and ferulate, ( Figure 1D ), provided at concentrations between 25 and 100 mM. While the lower substrate concentrations resulted in the highest observed growth rates on the byproduct fractions of both substrates, with the cultures on 25 mM and 50 mM p -coumarate exceeding the cultures on 25 mM and 50 mM ferulate, at 100 mM no significant difference was observed ( Table 3 ). Growth on p -coumarate resulted in higher maximal optical densities under all tested conditions. It was anticipated that formaldehyde released from ferulate should lead to higher final biomass concentrations similar to the results of co-feeding glucose and formate to P. putida KT2440 ( Zobel et al ., 2017 ). Quantification of Substrate Conversion to Protocatechuate The observed growth behaviour of the deletion mutant on ferulate indicated a limitation in either formaldehyde oxidation as reported for P. putida KT2440 grown on vanillate or in the demethylation of vanillate and hence formaldehyde release. Indeed, it was shown previously that P. putida ’s native vanillate O-demethylase (encoded by vanAB ) can constitute a bottleneck in ferulate conversion ( Salvachúa et al ., 2018 ; Werner et al ., 2023 ). In both cases, formaldehyde cannot be used as an electron source. To test these hypotheses, by-product formation was determined in shake flask experiments with 75 mM p -coumarate or ferulate ( Figure 2 ). Under both conditions, the aromatic acid precursors of protocatechuate, p -hydroxybenzoate and vanillate, accumulated, as reported by Werner et al . (2023) . Accumulation of vanillate in the ferulate conditions limited the release of formaldehyde and NADH generation from its oxidation, explaining the absence of a growth advantage over p -coumarate. Furthermore, we observed the accumulation of p -hydroxybenzoate in the p -coumarate condition, which could be attributed to the beneficial NADH savings suggested by the in silico results. No accumulation of the aldehyde products ( p -hydroxybenzaldehyde/vanillin) was observed. Download figure Open in new tab Figure 2: A Pathway for protocatechuate production from p -coumarate/ferulate in the Δ pcaHG mutant. The detected compounds ferulate/ p -coumarate, vanillate/ p -hydroxybenzoate, and protocatechuate are highlighted in orange, light blue, and dark blue, respectively. B Concentration profile of p -coumarate, p -hydroxybenzoate, and protocatechuate of the Δ pcaHG mutant grown on p -coumarate. C Concentration profile of ferulate, vanillin, and protocatechuate of the Δ pcaHG mutant grown on ferulate. Aromatic compound concentrations are shown as dashed lines, and the measured OD 600 is included as solid line. The standard deviation of triplicates is indicated by the shaded area. Overexpressing the native pobA and vanAB genes or their homologs, can support p- hydroxybenzoate and vanillate conversion ( Kuatsjah et al ., 2022 ; Werner et al ., 2023 ), with the latter leading to formaldehyde release. P. putida possesses a set of formaldehyde and formate dehydrogenases with high activities ( Roca et al ., 2009 ; Turlin et al ., 2023 ). Despite its arsenal of formaldehyde and formate dehydrogenases constitutively or conditionally expressed to prevent toxicity ( Roca & Ramos, 2009 ; Turlin et al., 2023 ), formaldehyde accumulation was observed during growth on vanillate ( Lee et al., 2021 , 2022 ). To observe the beneficial effects of formaldehyde oxidation predicted by metabolic modelling, stronger constitutive expression of formaldehyde and formate dehydrogenases might be necessary. Product yields were compared under both conditions to assess overall conversion efficiency. Only 32.3±0.8 % of p -coumarate and 7.4±0.2 % of ferulate were converted to protocatechuate. The rest accumulated as p -hydroxybenzoate (77.0±0.8 %) and vanillate (82.6±2.0 %), while 2.7±0.0 % of p -coumarate and 13.6±0.1 % ferulate remained in the media at the end of the cultivation. The sum of the final concentrations of protocatechuate and the respective aromatic acid precursor indicated a molar yield of approx. 100 % in the case of p -coumarate and 90.0±2.2 % for ferulate. Previous studies reported molar yields of 93±1 % and 94±7 % for ketoadipate production from p -coumarate/ferulate ( Werner et al ., 2023 ) but supplemented additional carbon sources like glucose or acetate for biomass formation ( Becker et al ., 2018 ; Johnson et al ., 2017 ; Werner et al ., 2023 ). When accounting for this additional carbon, the overall carbon yield was only 52.6 % in the case of the study of Werner et al . (2023) . In comparison, we achieved a carbon yield of 78 % for the sum of products, confirming that the C2 by-product fraction alone is sufficient for maintaining the growth of lignin valorisation strains while increasing the carbon efficiency. Indeed, the total product yield equals the theoretical maximum carbon yield for p -coumarate. In silico Study of C1 Assimilation Pathways for Application in Lignin Valorisation To lower CO 2 emissions from formaldehyde oxidation and increase the carbon available for growth, integration of C1 assimilation pathways into lignin valorisation strains can be considered. Indeed, overexpression of key enzymes of the ribulose-5-monophosphate pathway in lignin-degrading bacteria with native 3-hexulose-6-phosphate-synthase and the 6-phosphate-3-hexulose-isomerase, has improved growth on vanillate ( Mitsui et al ., 2003 ). Including C1 assimilation pathways may also aid the growth of the Δ pcaHG mutant. Recent advances in heterologous expression of C1 assimilation pathways in heterotrophic microorganisms demonstrate its feasibility ( Bruinsma et al ., 2023 ; He et al ., 2020 , 2020 ; Keller et al ., 2022 ; Lu et al ., 2019 ; Turlin et al ., 2022 ; Wenk et al ., 2022 ). To study the effect of C1 assimilation pathways on lignin valorisation with P. putida , we compared different C1 assimilation pathways in silico , the synthetic acetyl-CoA (SACA) pathway, ribulose-5-monophosphate (RuMP) cycle, reductive glycine (RG) pathway, and the homoserine (HOM) cycle (see Figure 3 ). These pathways were chosen based on their different architecture, different co-factor demands, and the different entry points of their output metabolites into the central carbon metabolism. Simulations were carried out with the curated genome-scale model i JN1463 in COBRApy extended with the reactions for the C1 assimilation pathways. The sole carbon source for the simulations was ferulate, set at an uptake rate of 4.5 mmol · g CDW -1 · h -1 . Simulations with p -coumarate were included for comparison. Download figure Open in new tab Figure 3: C1 assimilation pathways for the in silico study on the application of C1 assimilation in lignin valorisation system: A homoserine (HOM) cycle ( He et al ., 2020 ); B ribulose-5-monophosphate (RuMP) cycle ( Bar-Even et al ., 2013 ); C synthetic acetyl-CoA (SACA) pathway ( Lu et al ., 2019 ); D reductive glycine (RG) pathway ( Bar-Even et al ., 2013 ; Bruinsma et al ., 2023 ; Turlin et al ., 2022 ). Metabolites and co-factors – fald: formaldehyde, gcald: glycolaldehyde, acp: acetyl-phosphate, accoa: acetyl-CoA, ru5p: ribulose-5-monophosphate, h6p: hexulose-6-phosphate, f6p: fructose-6-phosphate, pyr: pyruvate, g6p: glucose-6-phosphate, 6pgl: 6-phosphoglucono-1,5-lactone, 6pgc: 6-phosphogluconate, for: formate, 10fthf: 10-formyl-THF, methf: 5,10-methenyl-THF, mlthf: 5,10-methylene-THF, gly: glycine, ser: serine, hob: 4-hydroxy-2-oxobutanoate, hom: homoserine, phom: phosphohomoserine, thr: threonine, acald: acetaldehyde, THF: tetrahydrofolate. Enzymes – GALS: glycolaldehyde synthetase, ACPS: acetylphosphate synthetase, H6PS: hexulose-6-phosphate synthetase, H6PI: hexulose-6-phosphate isomerase, FTF: formate-THF ligase, GCS: glycine cleavage system, SAL: serine aldolase, HAL: 4-hydroxy-2-oxobutanoate aldolase, HAT: 4-hydroxy-2-oxobutanoate aminotransferase, All simulations suggested that assimilation of formaldehyde is beneficial for the growth of the lignin valorisation strain, with the RuMP cycle enabling the highest improvement of the biomass formation rate of 11 % ( Table 4 ). While the SACA pathway and the RuMP pathway assimilated all formaldehyde, the CO 2 emission rates indicated that the assimilated carbon was oxidised in the central carbon metabolism. Overall, the benefit of C1 assimilation was low due to the energy demand of the C1 assimilation. View this table: View inline View popup Download powerpoint Table 4: Simulation of growth of a protocatechuate production model (Δ pcaHG ) on the C2 and C1 by-product fractions from ferulate using different C1 assimilation pathways. The results for the Δ pcaHG model with p -coumarate ( p CA) and ferulate are included for comparison. Biomass formation rate, percentage of not assimilated, fully oxidised C1 fraction (C1 ox ), and CO 2 emission rate are listed. SACA: synthetic acetyl-CoA pathway; RuMP: ribulose-5-monophosphate cycle; RG: reductive glycine pathway; HOM: homoserine cycle. C1 assimilation pathways are highly energy demanding. Thus, photo-/chemoautotroph microbes that natively use these pathways rely on additional energy sources, such as light, hydrogen, phosphite, and auxiliary C1 co-substrates or reduced C1 sources like carbon monoxide ( Claassens et al ., 2018 ; Figueroa et al ., 2018 ). While sacrificial C1 compounds have been shown to be promising electron donors for heterotrophic microbes ( Turlin et al ., 2022 ; Zobel et al ., 2017 ), the aim of this study was to increase the carbon yield of the proposed lignin valorisation strain design. Therefore, we decided to analyse the effect of including a carbon-free electron carrier in the models that enables electron supply from, e.g., hydrogen or phosphite on fuelling formaldehyde assimilation. Hydrogen is the global focus for several different applications, and research and innovation for sustainable production of green hydrogen as an energy carrier is vigorously pursued. Heterologous expression of an oxygen-tolerant hydrogenase from Cupriavidus necator enabled hydrogen oxidation in P. putida and the augmented redox power was shown to improve the NADH-dependent biotransformation of n -octane to 1-octanol ( Lonsdale et al ., 2015 ). Similar to hydrogen, phosphite can be produced sustainably ( Zhai et al ., 2022 ), and it can serve as phosphate source in P. putida when expressing a phosphite dehydrogenase, providing one molecule of NADH per molecule of phosphite ( Asin-Garcia et al ., 2022 ). Furthermore, phosphite has a very low reduction level (-650 mV), making the phosphite dehydrogenase an attractive candidate for heterologous carbon-free NADH regeneration ( Claassens et al ., 2018 ; Costas et al ., 2001 ). The results of the simulations were identical for hydrogen and phosphite as electron donors and are detailed for hydrogen supply in Table 5 . However, in the case of phosphite, substantial phosphate export was observed due to a up to 100-fold higher demand for NADH than for phosphate. View this table: View inline View popup Download powerpoint Table 5: Simulation of growth on the C2 and C1 by-product fractions from ferulate of a protocatechuate production model (Δ pcaHG ) complemented with different C1 assimilation pathways and a hydrogenase reaction. The results for the Δ pcaHG model with p -coumarate ( p CA) and ferulate are included for comparison. Biomass formation rate, percentage of not assimilated, fully oxidised C1 fraction (C1 ox ), and CO 2 emission rate are listed. SACA: synthetic acetyl-CoA pathway; RuMP: ribulose-5-monophosphate cycle; RG: reductive glycine pathway; HOM: homoserine cycle. The utilisation of the inorganic electron source abolished formaldehyde oxidation in all models augmented with C1 assimilation pathways. CO 2 emission rates were reduced by 54 % and 72 % for the SACA pathway and RuMP cycle, respectively. In the case of the RG pathway and the HOM cycle, the simulations suggested CO 2 utilisation leading to net CO 2 uptake instead of CO 2 production. For the model incorporating the RG pathway, CO 2 assimilation was based on the reductive glycine complex, which fixes one molecule of CO 2 per molecule of glycine formed. Within the HOM cycle, increased fluxes through the pyruvate carboxylase led to CO 2 assimilation, whereby one molecule of CO 2 is condensed onto pyruvate to generate one molecule of oxaloacetate. These in silico results demonstrate potential benefits of co-assimilating lignin-derived formaldehyde with C2 by-product fractions from lignin aromatic monomers to maximally conserve the monomeric lignin substrates into the desired product. Model-Based Identification of a Novel C1 Assimilation Pathway for Growth-Coupled Lignin Valorisation The simulations involving the HOM cycle relied on the activity of the pyruvate carboxylase, an enzyme that was shown to carry significant flux in P. putida ( Zobel et al ., 2017 ). In addition, the simulations revealed that key reactions of the HOM cycle, namely the 4-hydroxy-2-oxobutanoate aldolase and aminotransferase reactions were inactive. Instead, the model used an alternative C1 assimilation pathway. This novel pathway is a hybrid that utilises the serine aldolase (SAL) reaction from the HOM cycle to condense formaldehyde to glycine, generating serine, and integrates parts of the serine threonine (ST) cycle, a formate assimilation pathway described by Wenk et al . (2022) (see Figure 4A ). In the ST cycle, formate is conjugated to tetrahydrofolate (THF) to form methylene-THF, which transfer the C1 moiety onto glycine, producing serine. Deamination of serine yields pyruvate, the substrate for pyruvate carboxylase, which fixes CO 2 -derived bicarbonate (HCO 3 - ) resulting in oxaloacetate. Oxaloacetate is further converted to threonine, which when cleaved by threonine aldolase, regenerates glycine, thereby closing the C1 assimilation pathway, and releases the C2 product acetaldehyde. Except for the assimilation of formate, all reactions of the ST cycle are native to P. putida , while serine aldolase activity has been shown to be functional in P. putida with the threonine aldolase native to Escherichia coli ( Puiggené et al ., 2025 ). Introducing a single gene encoding a serine aldolase together with the NADH regeneration module (hydrogenase/phosphite operon) in P. putida would enable formaldehyde co-assimilation via this novel C1 assimilation pathway, subsequently referred to as SAL shunt. Download figure Open in new tab Figure 4: Novel C1 assimilation pathways proposed in a protocatechuate-producing strain, enabling capture of the C1 by-product fraction from G lignin monomers (vanillin, vanillate, ferulate); A via the serine aldolase (SAL) shunt; B via the reductive glycine pathway-serine aldolase hybrid cycle (RGSAL). Considering the improvement in simulated biomass formation and the dependence on a single non-native reaction for formaldehyde assimilation while also fixing CO 2 , the SAL shunt was identified to be the most suitable C1 assimilation pathway among the tested pathways for the application in lignin valorisation strains solely relying on the C1 and C2 by-product fractions for growth. In addition, we performed a competitive FBA with a model including reactions of all tested C1 assimilation pathways to identify other potential combinations of C1 assimilating reactions, similar to the SAL shunt. The model suggested a pathway combining the SAL shunt with the RG pathway (see Figure 4B ). This pathway assimilates formaldehyde and CO 2 in a circular fashion. First formaldehyde is assimilated via the serine aldolase generating serine. A smaller fraction of serine is deaminated to pyruvate, which then captures CO 2 via the pyruvate carboxylase as in the SAL shunt; serine is cleaved via the reversible glycine hydroxymethyl transferase (encoded by glyA I-II) into glycine and 5,10-methylene-THF. The latter serves as a substrate for the reductive glycine complex of the RG pathway capturing one molecule of CO 2 and producing glycine. The pathway has thus three streams for glycine generation: 1) via serine cleavage by glycine hydroxymethyl transferase activity, 2) via the reductive glycine complex, and 3) via threonine cleavage catalysed by the threonine aldolase . While no benefit in terms of biomass formation was computed, this pathway has a significantly reduced NADH demand in comparison to the SAL shunt and the RG pathway. The required NADH per assimilated carbon atom is 1.64 for the SAL shunt and 0.52 for the RG pathway. The RGSAL cycle requires only 0.33 NADH per assimilated carbon atom. In all cases, growth of the lignin valorisation strain would be coupled to C1 and C2 by-product formation and therefore to the production of protocatechuate from methoxylated lignin monomers. Conclusion This study demonstrates a novel approach to lignin valorisation, highlighting the feasibility of utilising C2 (acetyl-CoA) by-products from p -coumarate and ferulate degradation for growth while conserving the valuable aromatic core of lignin monomers in the target product. Using a P. putida EM42 pcaHG deletion mutant, a near-perfect molar yield of approximately 100% was achieved for ferulate and p -coumarate conversion with 78% of the substrate carbon conserved in the aromatic degradation products. This represents the highest reported carbon yield for biotechnological lignin utilization to date. Our in silico analyses showed limited potential for capturing formaldehyde) from methoxylated lignin monomers as additional carbon source due to energy limitation. However, when enabling the utilisation of electron donors such as hydrogen or phosphite, simulations not only identified novel C1 assimilation pathway variants but also suggested a significant boost in carbon efficiency and reduced carbon loss as CO 2 . In conclusion, our study suggests a promising glucose-free lignin valorisation strategy solely relying on C1 and C2 by-product co-assimilation for growth enabled by synthetic C1 assimilation pathways. Funding This study was partially supported by the United States Army International Technology Center-Pacific (ITC-PAC; Contract No. FA520923C0013). D.B. is grateful for the funding provided by CSIRO as part of the CSIRO Postgraduate Top-Up Scholarship of the Advanced Engineering Biology Future Science Platform. P.I.N. gratefully acknowledges financial support from The Novo Nordisk Foundation through grants NNF20CC0035580, LiFe (NNF18OC0034818), TARGET (NNF21OC0067996), FM· Pseudomonas (NNF24OC0091501), and NovoF (NNF23OC0083631), and the European Union’s Horizon2020 Research and Innovation Programme under grant agreements No. 814418 ( SinFonia ) and No. 101082049 ( TOLERATE ). Acknowledgments The authors would like to thank the other members of the Systems Environmental Microbiology group at DTU Biosustain (Denmark) for their support and guidance in genetic engineering of Pseudomonas putida and their expertise in C1 assimilation pathways provided. The authors thank Dr Gabi Netzel of the Queensland Node of Metabolomics and Proteomics Australia for their support and assistance in this work. Q-MAP is supported by Bioplatforms Australia, an NCRIS funded initiative. References ↵ Almqvist , H. , Veras , H. , Li , K. , Garcia Hidalgo , J. , Hulteberg , C. , Gorwa-Grauslund , M. , Skorupa Parachin , N. , & Carlquist , M . ( 2021 ). Muconic Acid Production Using Engineered Pseudomonas putida KT2440 and a Guaiacol-Rich Fraction Derived from Kraft Lignin . ACS Sustainable Chemistry & Engineering , 9 ( 24 ), 8097 – 8106 . doi: 10.1021/acssuschemeng.1c00933 OpenUrl CrossRef ↵ Asin-Garcia , E. , Batianis , C. , Li , Y. , Fawcett , J. D. , de Jong , I. , & dos Santos , V. A. P. M. ( 2022 ). Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida . Microbial Cell Factories , 21 ( 1 ), 156 . doi: 10.1186/s12934-022-01883-5 OpenUrl CrossRef PubMed ↵ Bagdasarian , M. , Lurz , R. , Rückert , B. , Franklin , F. C. , Bagdasarian , M. M. , Frey , J. , & Timmis , K. N . ( 1981 ). Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas . Gene , 16 ( 1–3 ), 237 – 247 . doi: 10.1016/0378-1119(81)90080-9 OpenUrl CrossRef PubMed Web of Science ↵ Bar-Even , A. , Noor , E. , Flamholz , A. , & Milo , R . ( 2013 ). Design and analysis of metabolic pathways supporting formatotrophic growth for electricity-dependent cultivation of microbes . Biochimica et Biophysica Acta (BBA) - Bioenergetics , 1827 ( 8–9 ), 1039 – 1047 . doi: 10.1016/j.bbabio.2012.10.013 OpenUrl CrossRef Web of Science ↵ Becker , J. , Kuhl , M. , Kohlstedt , M. , Starck , S. , & Wittmann , C . ( 2018 ). Metabolic engineering of Corynebacterium glutamicum for the production of cis, cis-muconic acid from lignin . Microbial Cell Factories , 17 ( 1 ), 115 . doi: 10.1186/s12934-018-0963-2 OpenUrl CrossRef ↵ Becker , J. , & Wittmann , C . ( 2019 ). A field of dreams: Lignin valorization into chemicals, materials, fuels, and health-care products . Biotechnology Advances , 37 ( 6 ), 107360 . doi: 10.1016/j.biotechadv.2019.02.016 OpenUrl CrossRef ↵ Bitinaite , J. , Rubino , M. , Varma , K. H. , Schildkraut , I. , Vaisvila , R. , & Vaiskunaite , R . ( 2007 ). USER TM friendly DNA engineering and cloning method by uracil excision . Nucleic Acids Research , 35 ( 6 ), 1992 – 2002 . doi: 10.1093/nar/gkm041 OpenUrl CrossRef PubMed Web of Science ↵ Blank , L. M. , Ionidis , G. , Ebert , B. E. , Bühler , B. , & Schmid , A . ( 2008 ). Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase: Metabolic response of P. putida to organic solvents . FEBS Journal , 275 ( 20 ), 5173 – 5190 . doi: 10.1111/j.1742-4658.2008.06648.x OpenUrl CrossRef PubMed ↵ Bruinsma , L. , Wenk , S. , Claassens , N. J. , & Martins dos Santos , V. A. P. ( 2023 ). Paving the way for synthetic C1—Metabolism in Pseudomonas putida through the reductive glycine pathway . Metabolic Engineering , 76 , 215 – 224 . doi: 10.1016/j.ymben.2023.02.004 OpenUrl CrossRef PubMed ↵ Calero , P. , Jensen , S. I. , Bojanovič , K. , Lennen , R. M. , Koza , A. , & Nielsen , A. T . ( 2018 ). Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida . Biotechnology and Bioengineering , 115 ( 3 ), 762 – 774 . doi: 10.1002/bit.26495 OpenUrl CrossRef ↵ Claassens , N. J. , Sánchez-Andrea , I. , Sousa , D. Z. , & Bar-Even , A . ( 2018 ). Towards sustainable feedstocks: A guide to electron donors for microbial carbon fixation . Current Opinion in Biotechnology , 50 , 195 – 205 . doi: 10.1016/j.copbio.2018.01.019 OpenUrl CrossRef PubMed ↵ Costas , A. M. G. , White , A. K. , & Metcalf , W. W . ( 2001 ). Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88*210 . Journal of Biological Chemistry , 276 ( 20 ), 17429 – 17436 . doi: 10.1074/jbc.M011764200 OpenUrl Abstract / FREE Full Text ↵ Ebert , B. E. , Kurth , F. , Grund , M. , Blank , L. M. , & Schmid , A . ( 2011 ). Response of Pseudomonas putida KT2440 to Increased NADH and ATP Demand . Applied and Environmental Microbiology , 77 ( 18 ), 6597 – 6605 . doi: 10.1128/AEM.05588-11 OpenUrl Abstract / FREE Full Text ↵ Ebrahim , A. , Lerman , J. A. , Palsson , B. O. , & Hyduke , D. R . ( 2013 ). COBRApy: COnstraints-Based Reconstruction and Analysis for Python . BMC Systems Biology , 7 ( 1 ), 74 . doi: 10.1186/1752-0509-7-74 OpenUrl CrossRef PubMed ↵ Fenster , J. A. , Werner , A. Z. , Tay , J. W. , Gillen , M. , Schirokauer , L. , Hill , N. C. , Watson , A. , Ramirez , K. J. , Johnson , C. W. , Beckham , G. T. , Cameron , J. C. , & Eckert , C. A . ( 2022 ). Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p -coumarate . Metabolic Engineering Communications , 15 , e00204 . doi: 10.1016/j.mec.2022.e00204 OpenUrl CrossRef ↵ Figueroa , I. A. , Barnum , T. P. , Somasekhar , P. Y. , Carlström , C. I. , Engelbrektson , A. L. , & Coates , J. D . ( 2018 ). Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway . Proceedings of the National Academy of Sciences , 115 ( 1 ), E92 – E101 . doi: 10.1073/pnas.1715549114 OpenUrl Abstract / FREE Full Text ↵ Hartmans , S. , Smits , J. P. , van der Werf , M. J. , Volkering , F. , & de Bont , J. A. M. ( 1989 ). Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X . Applied and Environmental Microbiology , 55 ( 11 ), 2850 – 2855 . doi: 10.1128/aem.55.11.2850-2855.1989 OpenUrl Abstract / FREE Full Text ↵ Harwood , C. S. , & Parales , R. E . ( 1996 ). THE β-KETOADIPATE PATHWAY AND THE BIOLOGY OF SELF-IDENTITY . Annual Review of Microbiology , 50 ( 1 ), 553 – 590 . doi: 10.1146/annurev.micro.50.1.553 OpenUrl CrossRef PubMed Web of Science ↵ He , H. , Höper , R. , Dodenhöft , M. , Marlière , P. , & Bar-Even , A . ( 2020 ). An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli . Metabolic Engineering , 60 , 1 – 13 . doi: 10.1016/j.ymben.2020.03.002 OpenUrl CrossRef PubMed ↵ Jiménez , J. I. , Miñambres , B. , García , J. L. , & Díaz , E . ( 2002 ). Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440 . Environmental Microbiology , 4 ( 12 ), 824 – 841 . doi: 10.1046/j.1462-2920.2002.00370.x OpenUrl CrossRef PubMed Web of Science ↵ Johnson , C. W. , Abraham , P. E. , Linger , J. G. , Khanna , P. , Hettich , R. L. , & Beckham , G. T . ( 2017 ). Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440 . Metabolic Engineering Communications , 5 , 19 – 25 . doi: 10.1016/j.meteno.2017.05.002 OpenUrl CrossRef PubMed ↵ Johnson , C. W. , Salvachúa , D. , Rorrer , N. A. , Black , B. A. , Vardon , D. R. , St. John , P. C. , Cleveland , N. S. , Dominick , G. , Elmore , J. R. , Grundl , N. , Khanna , P. , Martinez , C. R. , Michener , W. E. , Peterson , D. J. , Ramirez , K. J. , Singh , P. , VanderWall , T. A. , Wilson , A. N. , Yi , X. , … Beckham , G. T . ( 2019 ). Innovative Chemicals and Materials from Bacterial Aromatic Catabolic Pathways . Joule , 3 ( 6 ), 1523 – 1537 . doi: 10.1016/j.joule.2019.05.011 OpenUrl CrossRef ↵ Kamimura , N. , Takahashi , K. , Mori , K. , Araki , T. , Fujita , M. , Higuchi , Y. , & Masai , E . ( 2017 ). Bacterial catabolism of lignin-derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism . Environmental Microbiology Reports , 9 ( 6 ), 679 – 705 . doi: 10.1111/1758-2229.12597 OpenUrl CrossRef PubMed ↵ Keller , P. , Reiter , M. A. , Kiefer , P. , Gassler , T. , Hemmerle , L. , Christen , P. , Noor , E. , & Vorholt , J. A . ( 2022 ). Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle . Nature Communications , 13 ( 1 ), 5243 . doi: 10.1038/s41467-022-32744-9 OpenUrl CrossRef PubMed ↵ Klein , V. J. , Irla , M. , Gil López , M. , Brautaset , T. , & Fernandes Brito , L . ( 2022 ). Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy . Microorganisms , 10 ( 2 ), 220 . doi: 10.3390/microorganisms10020220 OpenUrl CrossRef ↵ Kohlstedt , M. , Starck , S. , Barton , N. , Stolzenberger , J. , Selzer , M. , Mehlmann , K. , Schneider , R. , Pleissner , D. , Rinkel , J. , Dickschat , J. S. , Venus , J. , B.J.H. van Duuren , J. , & Wittmann , C. ( 2018 ). From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida . Metabolic Engineering , 47 , 279 – 293 . doi: 10.1016/j.ymben.2018.03.003 OpenUrl CrossRef PubMed ↵ Kuatsjah , E. , Johnson , C. W. , Salvachúa , D. , Werner , A. Z. , Zahn , M. , Szostkiewicz , C. J. , Singer , C. A. , Dominick , G. , Okekeogbu , I. , Haugen , S. J. , Woodworth , S. P. , Ramirez , K. J. , Giannone , R. J. , Hettich , R. L. , McGeehan , J. E. , & Beckham , G. T . ( 2022 ). Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate . Metabolic Engineering , 70 , 31 – 42 . doi: 10.1016/j.ymben.2021.12.010 OpenUrl CrossRef PubMed ↵ Lee , J. A. , Baugh , A. C. , Shevalier , N. J. , Strand , B. , Stolyar , S. , & Marx , C. J . ( 2021 ). Cross-Feeding of a Toxic Metabolite in a Synthetic Lignocellulose-Degrading Microbial Community . Microorganisms , 9 ( 2 ), Article 2. doi: 10.3390/microorganisms9020321 OpenUrl CrossRef ↵ Lee , J. A. , Stolyar , S. , & Marx , C. J . ( 2022 ). Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae . Frontiers in Microbiology , 13 . https://www.frontiersin.org/articles/10.3389/fmicb.2022.849573 ↵ Linger , J. G. , Vardon , D. R. , Guarnieri , M. T. , Karp , E. M. , Hunsinger , G. B. , Franden , M. A. , Johnson , C. W. , Chupka , G. , Strathmann , T. J. , Pienkos , P. T. , & Beckham , G. T . ( 2014 ). Lignin valorization through integrated biological funneling and chemical catalysis . Proceedings of the National Academy of Sciences , 111 ( 33 ), 12013 – 12018 . doi: 10.1073/pnas.1410657111 OpenUrl Abstract / FREE Full Text ↵ Lonsdale , T. H. , Lauterbach , L. , Honda Malca , S. , Nestl , B. M. , Hauer , B. , & Lenz , O . ( 2015 ). H 2 -driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O 2 -tolerant hydrogenase and a P450 monooxygenase . Chemical Communications , 51 ( 90 ), 16173 – 16175 . doi: 10.1039/C5CC06078H OpenUrl CrossRef PubMed ↵ Lu , X. , Liu , Y. , Yang , Y. , Wang , S. , Wang , Q. , Wang , X. , Yan , Z. , Cheng , J. , Liu , C. , Yang , X. , Luo , H. , Yang , S. , Gou , J. , Ye , L. , Lu , L. , Zhang , Z. , Guo , Y. , Nie , Y. , Lin , J. , … Jiang , H . ( 2019 ). Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design . Nature Communications , 10 ( 1 ), Article 1. doi: 10.1038/s41467-019-09095-z OpenUrl CrossRef PubMed ↵ Martínez-García , E. , Nikel , P. I. , Aparicio , T. , & de Lorenzo , V. ( 2014 ). Pseudomonas 2.0: Genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression . ↵ Mitsui , R. , Kusano , Y. , Yurimoto , H. , Sakai , Y. , Kato , N. , & Tanaka , M . ( 2003 ). Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid . Applied and Environmental Microbiology , 69 ( 10 ), 6128 – 6132 . doi: 10.1128/AEM.69.10.6128-6132.2003 OpenUrl Abstract / FREE Full Text ↵ Mohamed , E. T. , Werner , A. Z. , Salvachúa , D. , Singer , C. A. , Szostkiewicz , K. , Rafael Jiménez-Díaz , M. , Eng , T. , Radi , M. S. , Simmons , B. A. , Mukhopadhyay , A. , Herrgård , M. J. , Singer , S. W. , Beckham , G. T. , & Feist , A. M. ( 2020 ). Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance . Metabolic Engineering Communications , 11 , e00143 . doi: 10.1016/j.mec.2020.e00143 OpenUrl CrossRef PubMed ↵ Neves , D. , Meinen , D. , Alter , T. B. , Blank , L. M. , & Ebert , B. E . ( 2024 ). Expanding Pseudomonas taiwanensis VLB120’s acyl-CoA portfolio: Propionate production in mineral salt medium . Microbial Biotechnology , 17 ( 1 ), e14309 . doi: 10.1111/1751-7915.14309 OpenUrl CrossRef ↵ Neves , D. , Vos , S. , Blank , L. M. , & Ebert , B. E . ( 2020 ). Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency . Frontiers in Bioengineering and Biotechnology , 7 . https://www.frontiersin.org/articles/10.3389/fbioe.2019.00458 ↵ Nikel , P. I. , Chavarría , M. , Danchin , A. , & de Lorenzo , V. ( 2016 ). From dirt to industrial applications: Pseudomonas putida as a Synthetic Biology chassis for hosting harsh biochemical reactions . Current Opinion in Chemical Biology , 34 , 20 – 29 . doi: 10.1016/j.cbpa.2016.05.011 OpenUrl CrossRef PubMed ↵ Nikel , P. I. , & de Lorenzo , V. ( 2018 ). Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans -metabolism . Metabolic Engineering , 50 , 142 – 155 . doi: 10.1016/j.ymben.2018.05.005 OpenUrl CrossRef ↵ Nikel , P. I. , Fuhrer , T. , Chavarría , M. , Sánchez-Pascuala , A. , Sauer , U. , & de Lorenzo , V. ( 2021 ). Reconfiguration of metabolic fluxes in Pseudomonas putida as a response to sub-lethal oxidative stress . The ISME Journal , 15 ( 6 ), 1751 – 1766 . doi: 10.1038/s41396-020-00884-9 OpenUrl CrossRef ↵ Nikel , P. I. , Martínez-García , E. , & de Lorenzo , V. ( 2014 ). Biotechnological domestication of pseudomonads using synthetic biology . Nature Reviews Microbiology , 12 ( 5 ), 368 – 379 . doi: 10.1038/nrmicro3253 OpenUrl CrossRef PubMed ↵ Nogales , J. , García , J. L. , & Díaz , E. ( 2017 ). Degradation of Aromatic Compounds in Pseudomonas: A Systems Biology View . In F. Rojo (Ed.), Aerobic Utilization of Hydrocarbons, Oils and Lipids (pp. 1 – 49 ). Springer International Publishing . doi: 10.1007/978-3-319-39782-5_32-1 OpenUrl CrossRef ↵ Nogales , J. , Mueller , J. , Gudmundsson , S. , Canalejo , F. J. , Duque , E. , Monk , J. , Feist , A. M. , Ramos , J. L. , Niu , W. , & Palsson , B. O . ( 2020 ). High-quality genome-scale metabolic modelling of Pseudomonas putida highlights its broad metabolic capabilities . Environmental Microbiology , 22 ( 1 ), 255 – 269 . doi: 10.1111/1462-2920.14843 OpenUrl CrossRef ↵ Notonier , S. , Werner , A. Z. , Kuatsjah , E. , Dumalo , L. , Abraham , P. E. , Hatmaker , E. A. , Hoyt , C. B. , Amore , A. , Ramirez , K. J. , Woodworth , S. P. , Klingeman , D. M. , Giannone , R. J. , Guss , A. M. , Hettich , R. L. , Eltis , L. D. , Johnson , C. W. , & Beckham , G. T . ( 2021 ). Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid . Metabolic Engineering , 65 , 111 – 122 . doi: 10.1016/j.ymben.2021.02.005 OpenUrl CrossRef PubMed Platt , R. , Drescher , C. , Park , S.-K. , & Phillips , G. J . ( 2000 ). Genetic System for Reversible Integration of DNA Constructs and lacZ Gene Fusions into the Escherichia coli Chromosome . Plasmid , 43 ( 1 ), 12 – 23 . doi: 10.1006/plas.1999.1433 OpenUrl CrossRef PubMed Web of Science ↵ Poblete-Castro , I. , Becker , J. , Dohnt , K. , dos Santos , V. M. , & Wittmann , C. ( 2012 ). Industrial biotechnology of Pseudomonas putida and related species . Applied Microbiology and Biotechnology , 93 ( 6 ), 2279 – 2290 . doi: 10.1007/s00253-012-3928-0 OpenUrl CrossRef PubMed Web of Science ↵ Puiggené , Ò. , Muñoz-Triviño , J. , Civil-Ferrer , L. , Gille , L. , Schulz-Mirbach , H. , Bergen , D. , Erb , T. J. , Ebert , B. E. , & Nikel , P. I . ( 2025 ). Systematic engineering of synthetic serine cycles in Pseudomonas putida uncovers emergent topologies for methanol assimilation . bioRxiv . ↵ Ragauskas , A. J. , Beckham , G. T. , Biddy , M. J. , Chandra , R. , Chen , F. , Davis , M. F. , Davison , B. H. , Dixon , R. A. , Gilna , P. , Keller , M. , Langan , P. , Naskar , A. K. , Saddler , J. N. , Tschaplinski , T. J. , Tuskan , G. A. , & Wyman , C. E . ( 2014 ). Lignin Valorization: Improving Lignin Processing in the Biorefinery . Science , 344 ( 6185 ), 1246843 . doi: 10.1126/science.1246843 OpenUrl Abstract / FREE Full Text ↵ Ravi , K. , García-Hidalgo , J. , Gorwa-Grauslund , M. F. , & Lidén , G . ( 2017 ). Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost . Applied Microbiology and Biotechnology , 101 ( 12 ), 5059 – 5070 . doi: 10.1007/s00253-017-8211-y OpenUrl CrossRef ↵ Rinaldi , R. , Jastrzebski , R. , Clough , M. T. , Ralph , J. , Kennema , M. , Bruijnincx , P. C. A. , & Weckhuysen , B. M . ( 2016 ). Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis . Angewandte Chemie International Edition , 55 ( 29 ), 8164 – 8215 . doi: 10.1002/anie.201510351 OpenUrl CrossRef PubMed ↵ Roca , A. , & Ramos , J. L . ( 2009 ). In vivo role of FdhD and FdmE in formate metabolism in Pseudomonas putida: Redundancy and expression in the stationary phase . Environmental Microbiology Reports , 1 ( 3 ), 208 – 213 . doi: 10.1111/j.1758-2229.2009.00032.x OpenUrl CrossRef PubMed ↵ Roca , A. , Rodríguez-Herva , J. J. , & Ramos , J. L . ( 2009 ). Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida . Journal of Bacteriology , 191 ( 10 ), 3367 – 3374 . doi: 10.1128/JB.00076-09 OpenUrl Abstract / FREE Full Text ↵ Rouches , E. , Gómez-Alvarez , H. , Majira , A. , Martín-Moldes , Z. , Nogales , J. , Díaz , E. , Bugg , T. D. H. , & Baumberger , S . ( 2021 ). Assessment strategy for bacterial lignin depolymerization: Kraft lignin and synthetic lignin bioconversion with Pseudomonas putida . Bioresource Technology Reports , 15 , 100742 . doi: 10.1016/j.biteb.2021.100742 OpenUrl CrossRef ↵ Salvachúa , D. , Johnson , C. W. , Singer , C. A. , Rohrer , H. , Peterson , D. J. , Black , B. A. , Knapp , A. , & Beckham , G. T . ( 2018 ). Bioprocess development for muconic acid production from aromatic compounds and lignin . Green Chemistry , 20 ( 21 ), 5007 – 5019 . doi: 10.1039/C8GC02519C OpenUrl CrossRef ↵ Shinoda , E. , Takahashi , K. , Abe , N. , Kamimura , N. , Sonoki , T. , & Masai , E . ( 2019 ). Isolation of a novel platform bacterium for lignin valorization and its application in glucose-free cis,cis-muconate production . Journal of Industrial Microbiology and Biotechnology , 46 ( 8 ), 1071 – 1080 . doi: 10.1007/s10295-019-02190-6 OpenUrl CrossRef PubMed ↵ Turlin , J. , Dronsella , B. , De Maria , A. , Lindner , S. N. , & Nikel , P. I. ( 2022 ). Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation . Metabolic Engineering , 74 , 191 – 205 . doi: 10.1016/j.ymben.2022.10.008 OpenUrl CrossRef PubMed ↵ Turlin , J. , Puiggené , Ò. , Donati , S. , Wirth , N. T. , & Nikel , P. I . ( 2023 ). Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses . mSystems , 0 ( 0 ), e00004 – 23 . doi: 10.1128/msystems.00004-23 OpenUrl CrossRef PubMed ↵ Volke , D. C. , Martino , R. A. , Kozaeva , E. , Smania , A. M. , & Nikel , P. I . ( 2022 ). Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing . Nature Communications , 13 ( 1 ), Article 1. doi: 10.1038/s41467-022-30780-z OpenUrl CrossRef ↵ Volke , D. C. , Wirth , N. T. , & Nikel , P. I . ( 2021 ). Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids . Bio-Protocol , 11 ( 4 ), e3917 . doi: 10.21769/BioProtoc.3917 OpenUrl CrossRef ↵ Weiland , F. , Kohlstedt , M. , & Wittmann , C . ( 2022 ). Guiding stars to the field of dreams: Metabolically engineered pathways and microbial platforms for a sustainable lignin-based industry . Metabolic Engineering , 71 , 13 – 41 . doi: 10.1016/j.ymben.2021.11.011 OpenUrl CrossRef PubMed ↵ Weimer , A. , Kohlstedt , M. , Volke , D. C. , Nikel , P. I. , & Wittmann , C . ( 2020 ). Industrial biotechnology of Pseudomonas putida: Advances and prospects . Applied Microbiology and Biotechnology , 104 ( 18 ), 7745 – 7766 . doi: 10.1007/s00253-020-10811-9 OpenUrl CrossRef ↵ Wenk , S. , Rainaldi , V. , He , H. , Schann , K. , Bouzon , M. , Döring , V. , Lindner , S. N. , & Bar-Even , A . ( 2022 ). Synthetic carbon fixation via the autocatalytic serine threonine cycle [Preprint] . Synthetic Biology . doi: 10.1101/2022.09.28.509898 OpenUrl CrossRef ↵ Werner , A. Z. , Cordell , W. T. , Lahive , C. W. , Klein , B. C. , Singer , C. A. , Tan , E. C. D. , Ingraham , M. A. , Ramirez , K. J. , Kim , D. H. , Pedersen , J. N. , Johnson , C. W. , Pfleger , B. F. , Beckham , G. T. , & Salvachúa , D . ( 2023 ). Lignin conversion to β-ketoadipic acid by Pseudomonas putida via metabolic engineering and bioprocess development . Science Advances , 9 ( 36 ), eadj0053 . doi: 10.1126/sciadv.adj0053 OpenUrl CrossRef PubMed ↵ Zhai , F. , Xin , T. , Geeson , M. B. , & Cummins , C. C . ( 2022 ). Sustainable Production of Reduced Phosphorus Compounds: Mechanochemical Hydride Phosphorylation Using Condensed Phosphates as a Route to Phosphite . ACS Central Science , 8 ( 3 ), 332 – 339 . doi: 10.1021/acscentsci.1c01381 OpenUrl CrossRef PubMed ↵ Zhang , Z. , Wang , Y. , Zheng , P. , & Sun , J . ( 2021 ). Promoting Lignin Valorization by Coping with Toxic C1 Byproducts . Trends in Biotechnology , 39 ( 4 ), 331 – 335 . doi: 10.1016/j.tibtech.2020.09.005 OpenUrl CrossRef PubMed ↵ Zobel , S. , Kuepper , J. , Ebert , B. , Wierckx , N. , & Blank , L. M . ( 2017 ). Metabolic response of Pseudomonas putida to increased NADH regeneration rates . Engineering in Life Sciences , 17 ( 1 ), 47 – 57 . doi: 10.1002/elsc.201600072 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 22, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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